Determination of binding constants by means of equilibrium shifting

Abstract

There are disclosed a method for determining binding constants between a substance or a substance mixture and a target, and a kit for carrying out the method according to the invention. The binding constants of the substance for the targets are determined by shifting the binding equilibrium. The concentrations of the immobilised and the dissolved target are varied and the affinities with the targets are determined by shifting the binding equilibria in the individual batches.

The invention claimed is: 
     
       1. A method for determining binding constants of a substance with respect to a dissolved target and an immobilized target, comprising:
 incubating a first sample of the substance with a target immobilized on a first solid particulate carrier in a first sample container that contains a buffer solution and a dissolved target, 
 incubating a second sample of the substance with the target immobilized on a second solid particulate carrier in a second sample container that contains buffer solution and dissolved target, 
 incubating a third sample of the substance with the target immobilized on a third solid particulate carrier in a third sample container that contains buffer solution and dissolved target, 
 incubating a fourth sample of the substance with the target immobilized on a fourth solid particulate carrier in a fourth sample container that contains buffer solution and dissolved target, 
 wherein the first sample and second sample contain different amounts of the immobilized target and a same amount of the dissolved target, and the third sample and the fourth sample contain same amounts of the immobilized target as the first sample and the second sample and contain a same amount of the dissolved target; 
 wherein the amount of the dissolved target in the third sample container and the fourth sample container differs from the amount of the dissolved target in the first sample container and the second sample container, and 
 wherein the first sample container, the second sample container, the third sample container and the fourth sample container during incubation contain the same volume of liquid phase, composed of buffer solution, dissolved target, and substance sample, 
 the method further comprising:
 separating the solid particulate carrier from the respective incubation batches, 
 measuring the APA concentration of the substance not bound to the immobilized target in the supernatant of the respective incubation batch of the first, second, third and fourth containers, 
 
 determining a binding constant of the substance with respect to the immobilized target and a binding constant of the substance with respect to the dissolved target based on the measured APA concentrations of the first, second, third and fourth containers; 
 wherein the binding constants are dissociation constants and determination of the dissociation constants is carried out in equation I for each of the first, second, third and fourth containers: 
 
       
         
           
             
               
                 
                   
                     APA 
                     = 
                     
                       
                         
                           c 
                           0 
                         
                         · 
                         
                           K 
                           D 
                           H 
                         
                         · 
                         
                           ( 
                           
                             
                               [ 
                               P 
                               ] 
                             
                             + 
                             
                               K 
                               D 
                               P 
                             
                           
                           ) 
                         
                       
                       
                         
                           
                             [ 
                             immoT 
                             ] 
                           
                           · 
                           
                             K 
                             D 
                             P 
                           
                         
                         + 
                         
                           
                             K 
                             D 
                             H 
                           
                           · 
                           
                             ( 
                             
                               
                                 [ 
                                 P 
                                 ] 
                               
                               + 
                               
                                 K 
                                 D 
                                 P 
                               
                             
                             ) 
                           
                         
                       
                     
                   
                 
                 
                   
                     ( 
                     I 
                     ) 
                   
                 
               
             
           
         
         wherein 
         APA is a concentration of the substance that is not bound to the immobilized target, 
         K D   H  is dissociation constant of the immobilized target, 
         K D   P  is dissociation constant of the dissolved target, 
         c 0  is total constant added substance concentration, 
         [immoT ]is concentration of the immobilized target, and 
         [P ]is concentration of the dissolved target. 
       
     
     
       2. The method of  claim 1  wherein determining the dissociation constants further comprises:
 using a matrix of dissociation constants for K D   H  and K D   P  in equation I, 
 calculating respective APA concentrations to be expected for the matrix of dissociation constants, 
 comparing the calculated APA concentrations with the measured APA concentration, 
 selecting a value pair K D   H  and K D   P  that shows the smallest deviation between calculated APA concentration and measured APA concentration as specified dissociation constants of the substance to be tested with respect to the immobilized or the dissolved target. 
 
     
     
       3. The method of  claim 2  comprising selecting the value pair K D   H  and K D   P  that shows the smallest deviation between calculated APA concentration and measured APA concentration numerical optimization. 
     
     
       4. The method of  claim 1  further comprising incubating between one and twenty-one further samples with the target immobilized on a solid particulate carrier in at least one further sample container containing buffer solution and dissolved target, and preferably in respective further sample containers containing buffer solution,
 wherein at least one of the further samples is incubated in two sample containers with the same concentration of immobilized target, and at the same time, different concentrations of dissolved target, and 
 wherein all further sample containers contain the same amount of buffer solution during incubation as the first, second, third, and fourth sample containers. 
 
     
     
       5. The method of  claim 1  wherein the concentration of the substance (APA) in the supernatant of the respective incubation batch is determined relative to a reference sample. 
     
     
       6. The method of  claim 1  wherein the dissolved and the immobilized target are identical or different. 
     
     
       7. The method of  claim 1  wherein the carrier is insoluble in an aqueous solution. 
     
     
       8. The method of  claim 1  wherein the carrier is composed of an organic or inorganic polymer. 
     
     
       9. The method of  claim 1  wherein the carrier is in particulate form, wherein the particles are at least partially micro- or nanoscale particles. 
     
     
       10. The method of  claim 1  wherein the immobilized target is albumin. 
     
     
       11. The method of  claim 1  wherein the dissolved target is plasma or serum of human or animal origin. 
     
     
       12. The method of  claim 1  wherein separating the carrier comprises applying a magnetic field to detach the carrier. 
     
     
       13. The method of  claim 1  wherein measuring the concentration of the substance comprises at least one of mass spectrometry, fluorescence spectroscopy, methods using radioactivity, or chromatography methods or a combination of these methods. 
     
     
       14. The method of  claim 1  wherein the sample containers are cavities of microtiter plates or have surface characteristics that do not interfere with the substance. 
     
     
       15. The method of  claim 8  wherein the carrier is composed of agarose. 
     
     
       16. The method of  claim 1 , wherein:
 the first sample and second sample contain a first and a second amount of the immobilized target, respectively; 
 the first sample and second sample contain a third amount of the dissolved target, 
 the third sample and the fourth sample contain the first and the second amount of the immobilized target, respectively; 
 the third sample and the fourth sample contain a fourth amount of the dissolved target; 
 the first amount is different than the second amount; and 
 the third amount is different than the fourth amount.